U.S. patent number 6,573,052 [Application Number 09/877,095] was granted by the patent office on 2003-06-03 for method of determining a chemotherapeutic regimen based on ercci expression.
This patent grant is currently assigned to Response Genetics, Inc.. Invention is credited to Kathleen D. Danenberg.
United States Patent |
6,573,052 |
Danenberg |
June 3, 2003 |
Method of determining a chemotherapeutic regimen based on ERCCI
expression
Abstract
The present invention relates to prognostic methods which are
useful in medicine, particularly cancer chemotherapy. The object of
the invention to provide a method for assessing ERCC1 expression
levels in fixed or fixed and paraffin embedded tissues and
prognosticate the probable resistance of a patient's tumor to
treatment with platinum-based therapies by examination of the
amount of ERCC1 mRNA in a patient's tumor cells and comparing it to
a predetermined threshold expression level. More specifically, the
invention provides to oligonucleotide primer pair ERCC1 and methods
comprising their use for detecting levels of ERCC1 mRNA.
Inventors: |
Danenberg; Kathleen D.
(Altadena, CA) |
Assignee: |
Response Genetics, Inc. (New
York, NY)
|
Family
ID: |
27400285 |
Appl.
No.: |
09/877,095 |
Filed: |
June 11, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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796491 |
Mar 2, 2001 |
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Current U.S.
Class: |
435/6.12;
435/91.1; 435/91.2; 536/23.1; 536/24.33 |
Current CPC
Class: |
C12Q
1/6886 (20130101); Y10S 435/81 (20130101); C12Q
2600/106 (20130101); C12Q 2600/158 (20130101); C12Q
1/6841 (20130101) |
Current International
Class: |
A61K
33/24 (20060101); C07H 21/00 (20060101); C07H
21/04 (20060101); C12P 19/00 (20060101); C12P
19/34 (20060101); C12Q 1/68 (20060101); C12N
15/11 (20060101); C12Q 001/68 (); C12P 019/34 ();
C07H 021/04 () |
Field of
Search: |
;435/6,91.1,91.2
;536/23.1,24.33 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
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86, pp. 113-119. .
G. Stanta, et al., RNA Extraction from Formalin-Fixed and
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86, pp. 23-26. .
Iqbal S, Lenz HJ, "Determinants of prognosis and response to
therapy in colorectal cancer," Curr Oncol Rep. Mar.
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Quantitative RT-PCR Technique from Formalin-Fixed and
Paraffin-Embedded (FFPE) Tissues, Using the Thymidylate Synthase
(TS) Gene As a Target," American Society of Clinical Oncology: 17:
Abstract 2159, 1998. .
Salonga D, Danenberg KD, Johnson M, Metzger R, Groshen S, Tsao-Wei
Dd, Lenz HJ, Leichman CG, Leichman L, Diasio RB, Danenberg PV,
"Colorectal tumors responding to 5-fluorouracil have low gene
expression levels of dihydropyrimidine dehydrogenase, thymidylate
synthase, and thymidine phosphorylase," Clin Cancer Res. Apr.
2000;6(4):1322-7. .
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"Quantitative gene expression analysis in microdissected archival
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Heid, CA, "Real Time quantitative PCR," Genome Res 6: 986-994,
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270:467-470. .
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Expression", Gene (1995) vol. 156: 207-213. .
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E, "Increased mRNA levels of xeroderma pigmentosum complementation
group B (XPB) and Cockayne's syndrome complementation group B (CSB)
without increased mRNA levels of multidrug-resistance gene (MDR1)
or metallothionein-II (MT-II) in platinum-resistant human ovarian
cancer tissues." Biochem Pharmacol. Dec. 1, 2000;60(11):1611-9.
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Damia G, Guidi G, D'Incalci M, "Expression of genes involved in
nucleotide excision repair and sensitivity to cisplatin and
melphalan in human cancer cell lines," Eur J Cancer, Oct.
1998;34(11):1783-8. .
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Mangioni C, D'Incalci M, "Expression of genes of potential
importance in the response to chemotherapy and DNA repair in
patients with ovarian cancer," Gynecol Oncol. Apr. 1997;
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Hansson J., "Apoptosis and c-jun induction by cisplatin in a human
melanoma cell line and a drug-resistant daughter cell line,"
Anticancer Drug, Oct. 1995;6(5):657-68. .
Dabholkar M, Bostick-Bruton F, Weber C, Egwuagu C, Bohr VA, Reed
E., "Expression of excision repair genes in non-malignant bone
marrow from cancer patients," Mutat Res. Jan. 1993;293(2):151-60.
.
Dabholkar M, Bostick-Bruton F, Weber C, Bohr VA, Egwuagu C, Reed
E., "ERCC1 and ERCC2 expression in malignant tissues from ovarian
cancer patients," J Natl Cancer Inst. Oct. 7, 1992;84(19):1512-7.
.
Metzger R, et al., "ERCC1 mRNA Levels Complement Thymidylate
Synthase mRNA Levels in predicting response and survival for
gastric cancer patients receiving combination cisplatin and
fluorouracil chemotherapy," J Clin Oncol 16: 309-316, 1998. .
Taverna P, Hansson J, Scanlon KJ, Hill BT, "Gene expression in
X-irradiated human tumour cell lines expressing cisplatin
resistance and altered DNA repair capacity," Carcinogenesis. Sep.
1994;15(9):2053-6. .
Dabholkar M, Vionnet J, Bostick-Bruton F, Yu JJ, Reed E. Messenger
RNA levels of XPAC and ERCC1 in ovarian cancer tissue correlate
with response to platinum-based chemotherapy. J Clin Invest. Aug.
1994;94(2):703-8. .
Li Q, Yu JJ, Mu C, Yunmbam MK, Slavsky D, Cross CL, Bostick-Bruton
F, Reed E. Association between the level of ERCC-1 expression and
the repair of cisplatin-induced DNA damage in human ovarian cancer
cells. Anticancer Res. Mar.-Apr. 2000;20(2A):645-52. .
Johnston et al., TS Expression from Formalin Fixed Paraffin
Embedded (FFPE) tissues using Quantitative RT-PCR correlates with
frozen tissue data and predicts for response to 5-FU in metastatic
colorectal cancers. American Society of Clinical Oncology: Astract
2383, 1999..
|
Primary Examiner: Horlick; Kenneth R.
Assistant Examiner: Spiegler; Alexander H.
Attorney, Agent or Firm: Kenyon & Kenyon
Parent Case Text
This application is a Continuation-in-Part of U.S. Ser. No.
09/796,491, filed Mar. 2, 2001, and claims priority to Provisional
Patent Applications 60/250,121 filed Dec. 1, 2000 and 60/250,470
filed Dec. 4, 2000.
Claims
What is claimed is:
1. A method for determining the level of Excision Repair
Cross-Complementing gene (ERCC1) expression in a fixed paraffin
embedded tissue sample comprising: (a) deparaffinizing the tissue
sample to obtain a deparaffinized sample; (b) isolating mRNA from
the deparaffinized sample in the presence of an effective amount of
a chaotropic agent by first heating the tissue sample in a solution
comprising an effective concentration of a chatropic compound to a
temperature in the range of about 75 to about 100.degree. C. for a
time period of 5 to 120 minutes and recovering said mRNA from said
chaotropic solution; and (c) subjecting the mRNA to amplification
using a pair of oligonucleotide primers capable of amplifying a
region of the ERCC1 gene, to obtain an amplified sample; (d)
determining the quantity of ERCC1 mRNA relative to the quantity of
an internal control gene's mRNA.
2. The method of claim 1 wherein the pair of oligonucleotide
primers consists of SEQ ID NO: 1 or an oligonucleotide at least
about 80% identical thereto and SEQ ID NO: 2 or an oligonucleotide
primer at least about 80% identical thereto.
3. The method of claim 1 wherein, the internal control gene is
.beta.-actin.
4. A method for determining the level of Excision Repair
Cross-Complementing gene (ERCC1) expression in a fixed paraffin
embedded tissue sample comprising: a. deparaffinizing the tissue
sample to obtain a deparaffinized sample; b. isolating mRNA from
the deparaffinized sample by first heating the deparaffinized
tissue sample in a solution comprising an effective concentration
of a chaotropic agent to a temperature in the range of about 50 to
about 100.degree. C. and recovering said mRNA from said solution;
and c. determining the quantity of ERCC1 mRNA relative to the
quantity of an internal control gene's mRNA.
5. The method of claim 4, wherein the heating takes place from
about 5 to about 120 minutes.
Description
FIELD OF THE INVENTION
The present invention relates to prognostic methods which are
useful in medicine, particularly cancer chemotherapy. More
particularly, the invention relates to assessment of tumor cell
gene expression in a patient. The resistance of tumor cells to
chemotherapeutic agents that target DNA, especially agents that
damage DNA in the manner of platinating agents is assayed by
examining the mRNA expressed from genes involved in DNA repair in
humans.
BACKGROUND OF THE INVENTION
Cancer arises when a normal cell undergoes neoplastic
transformation and becomes a malignant cell. Transformed
(malignant) cells escape normal physiologic controls specifying
cell phenotype and restraining cell proliferation. Transformed
cells in an individual's body thus proliferate, forming a tumor.
When a tumor is found, the clinical objective is to destroy
malignant cells selectively while mitigating any harm caused to
normal cells in the individual undergoing treatment.
Chemotherapy is based on the use of drugs that are selectively
toxic (cytotoxic) to cancer cells. Several general classes of
chemotherapeutic drugs have been developed, including drugs that
interfere with nucleic acid synthesis, protein synthesis, and other
vital metabolic processes. These generally are referred to as
antimetabolite drugs. Other classes of chemotherapeutic drugs
inflict damage on cellular DNA. Drugs of these classes generally
are referred to as genotoxic. Susceptibility of an individual
neoplasm to a desired chemotherapeutic drug or combination of drugs
often, however, can be accurately assessed only after a trial
period of treatment. The time invested in an unsuccessful trial
period poses a significant risk in the clinical management of
aggressive malignancies.
The repair of damage to cellular DNA is an important biological
process carried out by a cell's enzymatic DNA repair machinery.
Unrepaired lesions in a cell's genome can impede DNA replication,
impair the replication fidelity of newly synthesized DNA and/or
hinder the expression of genes needed for cell survival. Thus,
genotoxic drugs generally are considered more toxic to actively
dividing cells that engage in DNA synthesis than to quiescent,
nondividing cells. Normal cells of many body tissues are quiescent
and commit infrequently to re-enter the cell cycle and divide.
Greater time between rounds of cell division generally is afforded
for the repair of DNA damage in normal cells inflicted by
chemotherapeutic genotoxins. As a result, some selectivity is
achieved for the killing of cancer cells. Many treatment regimens
reflect attempts to improve selectivity for cancer cells by
coadministering chemotherapeutic drugs belonging to two or more of
these general classes.
Because effective chemotherapy in solid tumors usually requires a
combination of agents, the identification and quantification of
determinants of resistance or sensitivity to each single drug has
become an important tool to design individual combination
chemotherapy.
Two widely used genotoxic anticancer drugs that have been shown to
damage cellular DNA are cisplatin (DDP) and carboplatin. Cisplatin
and/or carboplatin currently are used in the treatment of selected,
diverse neoplasms of epithelial and mesenchymal origin, including
carcinomas and sarcomas of the respiratory, gastrointestinal and
reproductive tracts, of the central nervous system, and of squamous
origin in the head and neck. Cisplatin in combination with other
agents is currently preferred for the management of testicular
carcinoma, and in many instances produces a lasting remission.
(Loehrer et al., 1984,100 Ann. Int. Med. 704). Cisplatin (DDP)
disrupts DNA structure through formation of intrastrand adducts.
Resistance to platinum agents such as DDP has been attributed to
enhanced tolerance to platinum adducts, decreased drug
accumulation, or enhanced DNA repair. Although resistance to DDP is
multifactoral, alterations in DNA repair mechanisms probably play a
significant role. Excision repair of bulky DNA adducts, such as
those formed by platinum agents, appears to be mediated by genes
involved in DNA damage recognition and excision. Cleaver et al.,
Carcinogenesis 11:875-882 (1990); Hoeijmakers et al., Cancer Cells
2:311-320 (1990); Shivji et al., Cell 69:367-374 (1992). Indeed,
cells carrying a genetic defect in one or more elements of the
enzymatic DNA repair machinery are extremely sensitive to
cisplatin. Fraval et al. (1978), 51 Mutat. Res. 121, Beck and
Brubaker (1973), 116 J. Bacteriol 1247.
The excision repair cross-complementing (ERCC1) gene is essential
in the repair of DNA adducts. The human ERCC1 gene has been cloned.
Westerveld et al., Nature (London) 310:425-428 (1984); Tanaka et
al., Nature 348:73-76 (1990). Several studies using mutant human
and hamster cell lines that are defective in this gene and studies
in human tumor tissues indicate that the product encoded by ERCC1
is involved in the excision repair of platinum-DNA adducts.
Dabholkar et al., J. Natl. Cancer Inst. 84:1512-1517 (1992); Dijt
et al., Cancer Res. 48:6058-6062 (1988); Hansson et al., Nucleic
Acids Res. 18: 35-40 (1990).
When transfected into DNA-repair deficient CHO cells, ERCC1 confers
cellular resistance to cisplatin along with the ability to repair
platinum-DNA adducts. Hansson et al., Nucleic Acids Res. 18: 35-40
(1990). Currently accepted models of excision repair suggest that
the damage recognition/excision step is rate-limiting to the
excision repair process.
The relative levels of expression of excision repair genes such as
ERCC1 in malignant cells from cancer patients receiving
platinum-based therapy has been examined. Dabholkar et al., J.
Natl. Cancer Inst. 84:1512-1517 (1992). ERCC1 overexpression in
gastric cancer patients has been reported to have a negative impact
on tumor response and ultimate survival when treated with the
chemotherapeutic regimen of cisplatin (DDP)/fluorouracil (Metzger,
et al., J Clin Oncol 16: 309, 1998). Recent evidence indicates that
gemcitabine (Gem) may modulate ERCC1 nucleotide excision repair
(NER) activity. Thus, intratumoral levels of ERCC1 expression may
be a major prognostic factor for determining whether or not DDP and
GEM would be an effective therapeutic cancer patients.
Most pathological samples are routinely fixed and paraffin-embedded
(FPE) to allow for histological analysis and subsequent archival
storage. Thus, most biopsy tissue samples are not useful for
analysis of gene expression because such studies require a high
integrity of RNA so that an accurate measure of gene expression can
be made. Currently, gene expression levels can be only
qualitatively monitored in such fixed and embedded samples by using
immunohistochemical staining to monitor protein expression
levels.
Until now, quantitative gene expression studies including those of
ERCC1 expression have been limited to reverse transcriptase
polymerase chain reaction (RT-PCR) amplification of RNA from fresh
or frozen tissue. U.S. Pat. No. 5,705,336 to Reed et al., discloses
a method of quantifying ERCC1 mRNA from ovarian tumor tissue and
determining whether that tissue will be sensitive to platinum-based
chemotherapy. Reed et al., quanitfy ERCC1 mRNA from frozen ovarian
tumor biopsies.
The use of frozen tissue by health care professionals as described
in Reed et al., poses substantial inconveniences. Rapid biopsy
delivery to avoid tissue and subsequent mRNA degradation is the
primary concern when planning any RNA-based quantitative genetic
marker assay. The health care professional performing the biopsy,
must hastily deliver the tissue sample to a facility equipped to
perform an RNA extraction protocol immediately upon tissue sample
receipt. If no such facility is available, the clinician must
promptly freeze the sample in order to prevent mRNA degradation. In
order for the diagnostic facility to perform a useful RNA
extraction protocol prior to tissue and RNA degradation, the tissue
sample must remain frozen until it reaches the diagnostic facility,
however far away that may be. Maintenance of frozen tissue
integrity during transport using specialized couriers equipped with
liquid nitrogen and dry ice, comes only at a great expense.
Routine biopsies generally comprise a heterogenous mix of stromal
and tumorous tissue. Unlike with fresh or frozen tissue, FPE biopsy
tissue samples are readily microdissected and separated into
stromal and tumor tissue and therefore, offer advantage over the
use of fresh or frozen tissue. However, isolation of RNA from fixed
tissue, and especially fixed and paraffin embedded tissue, results
in highly degraded RNA, which is generally not applicable to gene
expression studies.
A number of techniques exist for the purification of RNA from
biological samples, but none is reliable for isolation of RNA from
FPE samples. For example, Chomczynski (U.S. Pat. No. 5,346,994)
describes a method for purifying RNA from tissues based on a liquid
phase separation using phenol and guanidine isothiocyanate. A
biological sample is homogenized in an aqueous solution of phenol
and guanidine isothiocyanate and the homogenate thereafter mixed
with chloroform. Following centrifugation, the homogenate separates
into an organic phase, an interphase and an aqueous phase. Proteins
are sequestered in the organic phase, DNA in the interphase, and
RNA in the aqueous phase. RNA can be precipitated from the aqueous
phase. Unfortunately, this method is not applicable to fixed and
paraffin-embedded (FPE) tissue samples.
Other known techniques for isolating RNA typically utilize either
guanidine salts or phenol extraction, as described for example in
Sambrook, J. et al., (1989) at pp. 7.3-7.24, and in Ausubel, F. M.
et al., (1994) at pp. 4.0.3-4.4.7. Again, none of the known methods
provides reproducible quantitative results in the isolation of RNA
from paraffin-embedded tissue samples.
Techniques for the isolation of RNA from paraffin-embedded tissues
are thus particularly needed for the study of gene expression in
tumor tissues, since expression levels of certain receptors or
enzymes can be used to determine the likelihood of success of a
particular treatment.
There is a need for a method of quantifying ERCC1 mRNA from
paraffinized tissue in order to provide an early prognosis for
proposed genotoxic cancer therapies. As a result, there has been a
concerted yet unsuccessful effort in the art to obtain a
quantification of ERCC1 expression in fixed and paraffinized (FPE)
tissue. Accordingly, it is the object of the invention to provide a
method for assessing ERCC1 levels in tissues fixed and
paraffin-embedded (FPE) and prognosticate the probable resistance
of a patient's tumor to treatment with DNA damaging agents,
creating the type of lesions in DNA that are created by DNA
platinating agents, by examination of the amount of ERCC1 mRNA in a
patient's tumor cells and comparing it to a predetermined threshold
expression level.
SUMMARY OF THE INVENTION
In one aspect of the invention there is provided a method for
assessing levels of expression of ERCC1 mRNA obtained from fixed
and paraffin-embedded (FPE) fixed and paraffin-embedded (FPE) tumor
cells.
In another aspect of the invention there is provided a method of
quantifying the amount of ERCC1 mRNA expression relative to an
internal control from a fixed and paraffin-embedded (FPE) tissue
sample. This method includes isolation of total mRNA from said
sample and determining the quantity of ERCC1 mRNA relative to the
quantity of an internal control gene's mRNA.
In an embodiment of this aspect of the invention, there are
provided oligonucleotide primers having the sequence of ERCC1-504F
(SEQ ID NO: 1) or ERCC1-574R (SEQ ID NO:2) and sequences
substantially identical thereto. The invention also provides for
oligonucleotide primers having a sequence that hybridizes to SEQ ID
NO: 1 or SEQ ID NO:2 or their complements under stringent
conditions.
In yet another aspect of the invention there is provided a method
for determining a chemotherapeutic regimen for a patient,
comprising isolating RNA from a fixed and paraffin-embedded (FPE)
tumor sample; determining a gene expression level of ERCC1 in the
sample; comparing the ERCC1 gene expression levels in the sample
with a predetermined threshold level for the ERCC1 gene; and
determining a chemotherapeutic regimen based on results of the
comparison of the ERCC1 gene expression level with the
predetermined threshold level.
The invention further relates to a method of normalizing the
uncorrected gene expression (UGE) of ERCC1 relative to an internal
control gene in a tissue sample analyzed using TaqMan.RTM.
technology to known ERCC1 expression levels relative to an internal
control from samples analyzed by pre-TaqMan.RTM. technology.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the response of tumors to Cisplatin/Gem
treatment vs. ERCC1 gene expression in NSCLC. Patients responding
completely (CR) to Cisplatin/Gem treatment showed ERCC1 gene
expression levels below the 4.5.times.10.sup.-3 threshold value,
while most patients with progressive disease (PD) showed ERCC1 gene
expression levels above 4.5.times.10.sup.-3. Partial response (PR),
stable disease (SD).
FIG. 2 is a chart illustrating how to calculate ERCC1 expression
relative to an internal control gene. The chart contains data
obtained with two test samples, (unknowns 1 and 2), and illustrates
how to determine the uncorrected gene expression data (UGE). The
chart also illustrates how to normalize UGE generated by the
TaqMan.RTM. instrument with known relative ERCC1 values determined
by pre-TaqMan.RTM. technology. This is accomplished by multiplying
UGE to a correction factor K.sub.ERCC1. The internal control gene
in the figure is .beta.-actin and the calibrator RNA is Human Liver
Total RNA (Stratagene, Cat. #735017).
DETAILED DESCRIPTION OF THE INVENTION
The present invention resides in part in the finding that the
amount of ERCC1 mRNA is correlated with resistance to DNA
platinating agents. Tumors expressing high levels of ERCC1 mRNA are
considered likely to be resistant to platinum-based chemotherapy.
Conversely, those tumors expressing low amounts of ERCC1 mRNA are
likely to be sensitive to platinum-based chemotherapy. A patient's
relative expression of tumor ERCC1 mRNA is judged by comparing it
to a predetermined threshold expression level.
The invention relates to a method of quantifying the amount of
ERCC1 mRNA expression in fixed and paraffin-embedded (FPE) tissue
relative to gene expression of an internal control. The present
inventors have developed oligonucleotide primers that allow
accurate assessment of ERCC1 expression in tissues that have been
fixed and embedded. The invention oligonucleotide primers,
ERCC1-504F (SEQ ID NO: 1), ERCC1-574R (SEQ ID NO: 2), or
oligonucleotide primers substantially identical thereto, preferably
are used together with RNA extracted from fixed and paraffin
embedded (FPE) tumor samples. This measurement of ERCC1 gene
expression may then be used for prognosis of platinum-based
chemotherapy.
This embodiment of the invention involves first, a method for
reliable extraction of RNA from an FPE sample and second,
determination of the content of ERCC1 mRNA in the sample by using a
pair of oligonucleotide primers, preferably oligionucleotide primer
pair ERCC1-504F (SEQ ID NO: 1) and ERCC1-574R (SEQ ID NO: 2), or
oligonucleotides substantially identical thereto, for carrying out
reverse transcriptase polymerase chain reaction. RNA is extracted
from the FPE cells by any of the methods for mRNA isolation from
such samples as described in U.S. patent application Ser. No.
09/469,338, filed Dec. 20, 1999, now U.S. Pat. No. 6,248,535, and
is hereby incorporated by reference in its entirety.
The present method can be applied to any type of tissue from a
patient. For examination of resistance of tumor tissue, it is
preferable to examine the tumor tissue. In a preferred embodiment,
a portion of normal tissue from the patient from which the tumor is
obtained, is also examined. Patients whose normal tissues are
expected to be resistant to platinum-based chemotherapeutic
compounds, i.e., show a high level of ERCC1 gene expression, but
whose tumors are expected to be sensitive to such compounds, i.e.,
show a low level of ERCC1 gene expression, may then be treated with
higher amounts of the chemotherapeutic composition.
The methods of the present invention can be applied over a wide
range of tumor types. This allows for the preparation of individual
"tumor expression profiles" whereby expression levels of ERCC1 are
determined in individual patient samples and response to various
chemotherapeutics is predicted. Preferably, the methods of the
invention are applied to solid tumors, most preferably Non-Small
Cell Lung Cancer (NSCLC) tumors. For application of some
embodiments of the invention to particular tumor types, it is
preferable to confirm the relationship of ERCC1 gene expression
levels to clinical resistance by compiling a dataset that enables
correlation of a particular ERCC1 expression and clinical
resistance to platinum-based chemotherapy.
A "predetermined threshold level", as defined herein, is a level of
ERCC1 expression above which it has been found that tumors are
likely to be resistant to a platinum-based chemotherapeutic
regimen. Expression levels below this threshold level are likely to
be found in tumors sensitive to platinum-based chemotherapeutic
regimen. The range of relative expression of ERCC1, expressed as a
ratio of ERCC1: .beta.-actin, among tumors responding to a
platinum-based chemotherapeutic regimen is less than about
4.5.times.10.sup.-3. Tumors that do not respond to a platinum-based
chemotherapeutic regimen have relative expression of
ERCC1:.beta.-actin ratio above about 4.5.times.10.sup.-3. FIG. 1.
However, the present invention is not limited to the use of
.beta.-actin as an internal control gene.
In performing the method of this embodiment of the present
invention, tumor cells are preferably isolated from the patient.
Solid or lymphoid tumors or portions thereof are surgically
resected from the patient or obtained by routine biopsy. RNA
isolated from frozen or fresh samples is extracted from the cells
by any of the methods typical in the art, for example, Sambrook,
Fischer and Maniatis, Molecular Cloning, a laboratory manual, (2nd
ed.), Cold Spring Harbor Laboratory Press, New York, (1989).
Preferably, care is taken to avoid degradation of the RNA during
the extraction process.
However, tissue obtained from the patient after biopsy is often
fixed, usually by formalin (formaldehyde) or gluteraldehyde, for
example, or by alcohol immersion. Fixed biological samples are
often dehydrated and embedded in paraffin or other solid supports
known to those of skill in the art. Non-embedded, fixed tissue may
also be used in the present methods. Such solid supports are
envisioned to be removable with organic solvents for example,
allowing for subsequent rehydration of preserved tissue.
RNA is extracted from the FPE cells by any of the methods as
described in U.S. patent application Ser. No. 09/469,338, filed
Dec. 20, 1999, now U.S. Pat. No. 6,248,535, which is hereby
incorporated by reference in its entirety. Fixed and
paraffin-embedded (FPE) tissue samples as described herein refers
to storable or archival tissue samples. RNA may be isolated from an
archival pathological sample or biopsy sample which is first
deparaffinized. An exemplary deparaffinization method involves
washing the paraffinized sample with an organic solvent, such as
xylene, for example. Deparaffinized samples can be rehydrated with
an aqueous solution of a lower alcohol. Suitable lower alcohols,
for example include, methanol, ethanol, propanols, and butanols.
Deparaffinized samples may be rehydrated with successive washes
with lower alcoholic solutions of decreasing concentration, for
example. Alternatively, the sample is simultaneously deparaffinized
and rehydrated. RNA is then extracted from the sample.
For RNA extraction, the fixed or fixed and deparaffinized samples
can be homogenized using mechanical, sonic or other means of
homogenization. Rehydrated samples may be homogenized in a solution
comprising a chaotropic agent, such as guanidinium thiocyanate
(also sold as guanidinium isothiocyanate). Homogenized samples are
heated to a temperature in the range of about 50 to about
100.degree. C. in a chaotropic solution, which contains an
effective amount of a chaotropic agent, such as a guanidinium
compound. A preferred chaotropic agent is guanidinium
thiocyanate.
An "effective concentration of chaotropic agent" is chosen such
that at an RNA is purified from a paraffin-embedded sample in an
amount of greater than about 10-fold that isolated in the absence
of a chaotropic agent. Chaotropic agents include: guanidinium
compounds, urea, formamide, potassium iodiode, potassium
thiocyantate and similar compounds. The preferred chaotropic agent
for the methods of the invention is a guanidinium compound, such as
guanidinium isothiocyanate (also sold as guanidinium thiocyanate)
and guanidinium hydrochloride. Many anionic counterions are useful,
and one of skill in the art can prepare many guanidinium salts with
such appropriate anions. The effective concentration of guanidinium
solution used in the invention generally has a concentration in the
range of about 1 to about 5 M with a preferred value of about 4 M.
If RNA is already in solution, the guanidinium solution may be of
higher concentration such that the final concentration achieved in
the sample is in the range of about 1 to about 5 M. The guanidinium
solution also is preferably buffered to a pH of about 3 to about 6,
more preferably about 4, with a suitable biochemical buffer such as
Tris-Cl. The chaotropic solution may also contain reducing agents,
such as dithiothreitol (DTT) and .beta.-mercaptoethanol (BME). The
chaotropic solution may also contain RNAse inhibitors.
Homogenized samples may be heated to a temperature in the range of
about 50 to about 100.degree. C. in a chaotropic solution, which
contains an effective amount of a chaotropic agent, such as a
guanidinium compound. A preferred chaotropic agent is guanidinium
thiocyanate.
RNA is then recovered from the solution by, for example, phenol
chloroform extraction, ion exchange chromatography or
size-exclusion chromatography. RNA may then be further purified
using the techniques of extraction, electrophoresis,
chromatography, precipitation or other suitable techniques.
The quantification of ERCC1 mRNA from purified total mRNA from
fresh, frozen or fixed is preferably carried out using
reverse-transcriptase polymerase chain reaction (RT-PCR) methods
common in the art, for example. Other methods of quantifying of
ERCC1 mRNA include for example, the use of molecular beacons and
other labeled probes useful in multiplex PCR. Additionally, the
present invention envisages the quantification of ERCC1 mRNA via
use of PCR-free systems employing, for example fluorescent labeled
probes similar to those of the Invader.RTM. Assay (Third Wave
Technologies, Inc.). Most preferably, quantification of ERCC1 cDNA
and an internal control or house keeping gene (e.g. .beta.-actin)
is done using a fluorescence based real-time detection method (ABI
PRISM 7700 or 7900 Sequence Detection System [TaqMan.RTM.], Applied
Biosystems, Foster City, Calif.) or similar system as described by
Heid et al., (Genome Res 1996;6:986-994) and Gibson et al.(Genome
Res 1996;6:995-1001). The output of the ABI 7700 (TaqMan.RTM.
Instrument) is expressed in Ct's or "cycle thresholds". With the
TaqMan.RTM. system, a highly expressed gene having a higher number
of target molecules in a sample generates a signal with fewer PCR
cycles (lower Ct) than a gene of lower relative expression with
fewer target molecules (higher Ct).
As used herein, a "house keeping" gene or "internal control" is
meant to include any constitutively or globally expressed gene
whose presence enables an assessment of ERCC1 mRNA levels. Such an
assessment comprises a determination of the overall constitutive
level of gene transcription and a control for variations in RNA
recovery. "House-keeping" genes or "internal controls" can include,
but are not limited to the cyclophilin gene, .beta.-actin gene, the
transferrin receptor gene, GAPDH gene, and the like. Most
preferably, the internal control gene is .beta.-actin gene as
described by Eads et al., Cancer Research 1999; 59:2302-2306.
A control for variations in RNA recovery requires the use of
"calibrator RNA." The "calibrator RNA" is intended to be any
available source of accurately pre-quantified control RNA.
Preferably, Human Liver Total RNA (Stratagene, Cat. #735017) is
used.
"Uncorrected Gene Expression (UGE)" as used herein refers to the
numeric output of ERCC1 expression relative to an internal control
gene generated by the TaqMan.RTM. instrument. The equation used to
determine UGE is shown in Example 3, and illustrated with sample
calculations in FIG. 2.
A further aspect of this invention provides a method to normalize
uncorrected gene expression (UGE) values acquired from the
TaqMan.RTM. instrument with "known relative gene expression" values
derived from non-TaqMan.RTM. technology. Preferably, the known
non-TaqMan.RTM. derived relative ERCC1:.beta.-actin expression
values are normalized with TaqMan.RTM. derived ERCC1 UGE values
from a tissue sample.
"Corrected Relative ERCC1 Expression" as used herein refers to
normalized ERCC1 expression whereby UGE is multiplied with a ERCC1
specific correction factor (K.sub.ERCC1), resulting in a value that
can be compared to a known range of ERCC1 expression levels
relative to an internal control gene. Example 3 and FIG. 2
illustrate these calculations in detail. These numerical values
allow the determination of whether or not the "Corrected Relative
ERCC1 Expression" of a particular sample falls above or below the
"predetermined threshold" level. The predetermined threshold level
of Corrected Relative ERCC1 Expression to .beta.-actin level is
about 4.5.times.10.sup.-3. K.sub.ERCC1 specific for ERCC1, the
internal control .beta.-actin and calibrator Human Liver Total RNA
(Stratagene, Cat. #735017), is 1.54.times.10.sup.-3.
"Known relative gene expression" values are derived from previously
analyzed tissue samples and are based on the ratio of the RT-PCR
signal of a target gene to a constitutively expressed internal
control gene (e.g. .beta.-Actin, GAPDH, etc.). Preferably such
tissue samples are formalin fixed and paraffin-embedded (FPE)
samples and RNA is extracted from them according to the protocol
described in Example 1 and in U.S. patent application Ser. No.
09/469,338, filed Dec. 20, 1999, now U.S. Pat. No. 6,248,535, which
is hereby incorporated by reference in its entirety. To quantify
gene expression relative to an internal control standard
quantitative RT-PCR technology known in the art is used.
Pre-TaqMan.RTM. technology PCR reactions are run for a fixed number
of cycles (i.e., 30) and endpoint values are reported for each
sample. These values are then reported as a ratio of ERCC1
expression to .beta.-actin expression. See U.S. Pat. No. 5,705,336
to Reed et al.
K.sub.ERCC.sub.1 may be determined for an internal control gene
other than .beta.-actin and/or a calibrator RNA different than
Human Liver Total RNA (Stratagene, Cat. #735017). To do so, one
must calibrate both the internal control gene and the calibrator
RNA to tissue samples for which ERCC1 expression levels relative to
that particular internal control gene have already been determined
(i.e., "known relative gene expression"). Preferably such tissue
samples are formalin fixed and paraffin-embedded (FPE) samples and
RNA is extracted from them according to the protocol described in
Example 1 and in U.S. patent application Ser. No. 09/469,338, filed
Dec. 20, 1999, now U.S. Pat. No. 6,248,535, which is hereby
incorporated by reference in its entirety. Such a determination can
be made using standard pre-TaqMan#, quantitative RT-PCR techniques
well known in the art. Upon such a determination, such samples have
"known relative gene expression" levels of ERCC1 useful in the
determining a new K.sub.ERCC1 specific for the new internal control
and/or calibrator RNA as described in Example 3.
The methods of the invention are applicable to a wide range of
tissue and tumor types and so can be used for assessment of
clinical treatment of a patient and as a diagnostic or prognostic
tool for a range of cancers including breast, head and neck, lung,
esophageal, colorectal, and others. In a preferred embodiment, the
present methods are applied to prognosis of Non-Small Cell Lung
Cancer (NSCLC).
Pre-chemotherapy treatment tumor biopsies are usually available
only as fixed paraffin embedded (FPE) tissues, generally containing
only a very small amount of heterogeneous tissue. Such FPE samples
are readily amenable to microdissection, so that ERCC1 gene
expression may be determined in tumor tissue uncontaminated with
stromal tissue. Additionally, comparisons can be made between
stromal and tumor tissue within a biopsy tissue sample, since such
samples often contain both types of tissues.
Generally, any oligonucleotide pair that flanks a region of ERCC1
gene may be used to carry out the methods of the invention. Primers
hybridizing under stringent conditions to a region of the ERCC1
gene for use in the present invention will amplify a product
between 20-1000 base pairs, preferably 50-100 base pairs, most
preferably less than 100 base pairs.
The invention provides specific oligonucleotide primers pairs and
oligonucleotide primers substantially identical thereto, that allow
particularly accurate assessment of ERCC1 expression in FPE
tissues. Preferable are oligonucleotide primers, ERCC1-504F (SEQ ID
NO: 1) and ERCC1 (SEQ ID NO: 2), (also referred to herein as the
oligonucleotide primer pair ERCC1) and oligonucleotide primers
substantially identical thereto. The oliogonucleotide primers
ERCC1-504F (SEQ ID NO: 1) and ERCC1, (SEQ ID NO: 2) have been shown
to be particularly effective for measuring ERCC1 mRNA levels using
RNA extracted from the FPE cells by any of the methods for mRNA
isolation, for example as described Example 1 and in U.S. patent
application Ser. No. 09/469,338, filed Dec. 20, 1999, now U.S. Pat.
No. 6,248,535, which is hereby incorporated by reference in its
entirety.
"Substantially identical" in the nucleic acid context as used
herein, means hybridization to a target under stringent conditions,
and also that the nucleic acid segments, or their complementary
strands, when compared, are the same when properly aligned, with
the appropriate nucleotide insertions and deletions, in at least
about 60% of the nucleotides, typically, at least about 70%, more
typically, at least about 80%, usually, at least about 90%, and
more usually, at least, about 95-98% of the nucleotides. Selective
hybridization exists when the hybridization is more selective than
total lack of specificity. See, Kanehisa, Nucleic Acids Res.,
12:203-213(1984)
This invention includes substantially identical oligonucleotides
that hybridize under stringent conditions (as defined herein) to
all or a portion of the oligonucleotide primer sequence of
ERCC1-504F (SEQ ID NO: 1), its complement or ERCC1-574R (SEQ ID NO:
2), or its complement.
Under stringent hybridization conditions, only highly
complementary, i.e., substantially similar nucleic acid sequences
hybridize. Preferably, such conditions prevent hybridization of
nucleic acids having 4 or more mismatches out of 20 contiguous
nucleotides, more preferably 2 or more mismatches out of 20
contiguous nucleotides, most preferably one or more mismatch out of
20 contiguous nucleotides.
The hybridizing portion of the nucleic acids is typically at least
10 (e.g., 15) nucleotides in length. The hybridizing portion of the
hybridizing nucleic acid is at least about 80%, preferably at least
about 95%, or most preferably about at least 98%, identical to the
sequence of a portion or all of oligonucleotide primer ERCC1-504F
(SEQ ID NO: 1), its complement or ERCC1-574R (SEQ ID NO: 2), or its
complement.
Hybridization of the oligonucleotide primer to a nucleic acid
sample under stringent conditions is defined below. Nucleic acid
duplex or hybrid stability is expressed as a melting temperature
(T.sub.m), which is the temperature at which the probe dissociates
from the target DNA. This melting temperature is used to define the
required stringency conditions. If sequences are to be identified
that are substantially identical to the probe, rather than
identical, then it is useful to first establish the lowest
temperature at which only homologous hybridization occurs with a
particular concentration of salt (e.g. SSC or SSPE). Then assuming
that 1% mismatching results in a 1.degree. C. decrease in T.sub.m,
the temperature of the final wash in the hybridization reaction is
reduced accordingly (for example, if sequences having >95%
identity with the probe are sought, the final wash temperature is
decrease by 5.degree. C.). In practice, the change in T.sub.m can
be between 0.5.degree. C. and 1.5.degree. C. per 1% mismatch.
Stringent conditions involve hybridizing at 68.degree. C. in
5.times.SSC/5.times.Denhart's solution/1.0% SDS, and washing in
0.2.times.SSC/0.1% SDS at room temperature. Moderately stringent
conditions include washing in 3.times.SSC at 42.degree. C. The
parameters of salt concentration and temperature be varied to
achieve optimal level of identity between the primer and the target
nucleic acid. Additional guidance regarding such conditions is
readily available in the art, for example, Sambrook, Fischer and
Maniatis, Molecular Cloning, a laboratory manual, (2nd ed.), Cold
Spring Harbor Laboratory Press, New York, (1989) and F. M. Ausubel
et al eds., Current Protocols in Molecular Biology, John Wiley and
Sons (1994).
Oligonucleotide primers disclosed herein are capable of allowing
accurate assessment of ERCC1 gene expression in a fixed or fixed
and paraffin embedded tissue, as well as frozen or fresh tissue.
This is despite the fact that RNA derived from FPE samples is more
fragmented relative to that of fresh or frozen tissue. Thus, the
methods of the invention are suitable for use in assaying ERCC1
expression levels in FPE tissue where previously there existed no
way to assay ERCC1 gene expression using fixed tissues.
Genotoxic agents are those that form persistent genomic lesions and
are preferred for use as chemotherapeutic agents in the clinical
management of cancer. The rate of cellular repair of
genotoxin-induced DNA damage, as well as the rate of cell growth
via the cell division cycle, affects the outcome of genotoxin
therapy. Unrepaired lesions in a cell's genome can impede DNA
replication, impair the replication fidelity of newly synthesized
DNA or hinder the expression of genes needed for cell survival.
Thus, one determinant of a genotoxic agent's cytotoxicity
(propensity for contributing to cell death) is the resistance of
genomic lesions formed therefrom to cellular repair. Genotoxic
agents that form persistent genomic lesions, e.g., lesions that
remain in the genome at least until the cell commits to the cell
cycle, generally are more effective cytotoxins than agents that
form transient, easily repaired genomic lesions.
A general class of genotoxic compounds that are used for treating
many cancers and that are affected by levels of ERCC1 expression
are DNA alkylating agents and DNA intercalating agents. Psoralens
are genotoxic compounds known to be useful in the
photochemotherapeutic treatment of cutaneous diseases such as
psoriasis, vitiligo, fungal infections and cutaneous T cell
lymphoma. Harrison's Principles of Internal Medicine, Part 2
Cardinal Manifestations of Disease, Ch. 60 (12th ed. 1991). Another
general class of genotoxic compounds, members of which can alkylate
or intercalate into DNA, includes synthetically and naturally
sourced antibiotics. Of particular interest herein are
antineoplastic antibiotics, which include but are not limited to
the following classes of compounds represented by: amsacrine;
actinomycin A, C, D (alternatively known as dactinomycin) or F
(alternatively KS4); azaserine; bleomycin; carminomycin
(carubicin), daunomycin (daunorubicin), or 14-hydroxydaunomycin
(adriamycin or doxorubicin); mitomycin A, B or C; mitoxantrone;
plicamycin (mithramycin); and the like.
Still another general class of genotoxic agents that are commonly
used and that alkylate DNA, are those that include the
haloethylnitrosoureas, especially the chloroethylnitrosoureas.
Representative members of this broad class include carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine and
streptozotocin. Haloethylnitrosourea first agents can be analogs or
derivatives of any of the foregoing representative compounds.
Yet another general class of genotoxic agents, members of which
alkylate DNA, includes the sulfur and nitrogen mustards. These
compounds damage DNA primarily by forming covalent adducts at the
N7 atom of guanine. Representative members of this broad class
include chlorambucil, cyclophosphamide, ifosfamide, melphalan,
mechloroethamine, novembicin, trofosfamide and the like.
Oligonucleotides or analogs thereof that interact covalently or
noncovalently with specific sequences in the genome of selected
cells can also be used as genotoxic agents, if it is desired to
select one or more predefined genomic targets as the locus of a
genomic lesion.
Another class of agents, members of which alkylate DNA, include the
ethylenimines and methylmelamines. These classes include
altretamine (hexamethylmelamine), triethylenephosphoramide (TEPA),
triethylenethiophosphoramide (ThioTEPA) and triethylenemelamine,
for example.
Additional classes of DNA alkylating agents include the alkyl
sulfonates, represented by busulfan; the azinidines, represented by
benzodepa; and others, represented by, e.g., mitoguazone,
mitoxantrone and procarbazine. Each of these classes includes
analogs and derivatives of the respective representative
compounds.
From the measurement of the amount of ERCC1 mRNA that is expressed
in the tumor, the skilled practitioner can make a prognosis
concerning clinical resistance of a tumor to a particular
genotoxin, preferably a platinum-based chemotherapy, or to a
chemotherapy inducing a similar type of DNA damage. Platinum-based
chemotherapies cause a "bulky adduct" of the DNA, wherein the
primary effect is to distort the three-dimensional conformation of
the double helix. Such compounds are meant to be administered
alone, or together with other chemotherapies such as gemcitabine
(Gem) or 5-Fluorouracil (5-FU).
Many compounds are commonly given with platinum-based chemotherapy
agents. For example, BEP (bleomycin, etoposide, cisplatin) is used
for testicular cancer, MVAC (methotrexate, vinblastine,
doxorubicin, cisplatin) is used for bladder cancer, MVP (mitomycin
C, vinblastine, cisplatin) is used for non-small cell lung cancer
treatment. Many studies have documented interactions between
platinum-containing agents. Therapeutic drug synergism, for
example, has been reported for many drugs potentially included in a
platinum based chemotherapy. A very short list of recent references
for this include the following: Okamoto et al., Urology 2001;
57:188-192.; Tanaka et al., Anticancer Research 2001; 21:313-315;
Slamon et al., Seminars in Oncology 2001; 28:13-19; Lidor et al.,
Journal of Clinical Investigation 1993; 92:2440-2447; Leopold et
al., NCI Monographs 1987;99-104; Ohta et al., Cancer Letters 2001;
162:39-48; van Moorsel et al., British Journal of Cancer 1999;
80:981-990.
Platinum-based genotoxic chemotherapies comprises heavy metal
coordination compounds which form covalent DNA adducts. Generally,
these heavy metal compounds bind covalently to DNA to form, in
pertinent part, cis-1,2-intrastrand dinucleotide adducts.
Generally, this class is represented by
cis-diamminedichloroplatinum (II) (cisplatin), and includes
cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II)
(carboplatin), cis-diammino-(1,2-cyclohexyl)dichloroplatinum(II),
and cis-(1,2-ethylenediammine)dichloroplatinum(II). Platinum first
agents include analogs or derivatives of any of the foregoing
representative compounds.
Tumors currently manageable by platinum coordination compounds
include testicular, endometrial, cervical, gastric, squamous cell,
adrenocortical and small cell lung carcinomas along with
medulloblastomas and neuroblastomas. Trans-Diamminedichloroplatinum
(II) (trans-DDP) is clinically useless owing, it is thought, to the
rapid repair of its DNA adducts. The use of trans-DDP as a
chemotherapeutic agent herein likely would provide a compound with
low toxicity in nonselected cells, and high relative toxicity in
selected cells. In a preferred embodiment, the platinum compound is
cisplatin.
The invention being thus described, practice of the invention is
illustrated by the experimental examples provided below. The
skilled practitioner will realize that the materials and methods
used in the illustrative examples can be modified in various ways.
Such modifications are considered to fall within the scope of the
present invention.
EXAMPLES
Example 1
RNA Isolation from FPE Tissue
RNA is extracted from paraffin-embedded tissue by the following
general procedure. A. Deparaffinization and hydration of sections:
(1) A portion of an approximately 10 .mu.M section is placed in a
1.5 mL plastic centrifuge tube. (2) 600 .mu.L, of xylene are added
and the mixture is shaken vigorously for about 10 minutes at room
temperature (roughly 20 to 25.degree. C.). (3) The sample is
centrifuged for about 7 minutes at room temperature at the maximum
speed of the bench top centrifuge (about 10-20,000.times.g). (4)
Steps 2 and 3 are repeated until the majority of paraffin has been
dissolved. Two or more times are normally required depending on the
amount of paraffin included in the original sample portion. (5) The
xylene solution is removed by vigorously shaking with a lower
alcohol, preferably with 100% ethanol (about 600 .mu.L) for about 3
minutes. (6) The tube is centrifuged for about 7 minutes as in step
(3). The supernatant is decanted and discarded. The pellet becomes
white. (7) Steps 5 and 6 are repeated with successively more dilute
ethanol solutions: first with about 95% ethanol, then with about
80% and finally with about 70% ethanol. (8) The sample is
centrifuged for 7 minutes at room temperature as in step (3). The
supernatant is discarded and the pellet is allowed to dry at room
temperature for about 5 minutes. B. RNA Isolation with
Phenol-Chloroform (1) 400 .mu.L guanidine isothiocyanate solution
including 0.5% sarcosine and 8 .mu.L dithiothreitol is added. (2)
The sample is then homogenized with a tissue homogenizer
(Ultra-Turrax, IKA-Works, Inc., Wilmington, N.C.) for about 2 to 3
minutes while gradually increasing the speed from low speed (speed
1) to high speed (speed 5). (3) The sample is then heated at about
95.degree. C. for about 5-20 minutes. It is preferable to pierce
the cap of the tube containing the sample with a fine gauge needle
before heating to 95.degree. C. Alternatively, the cap may be
affixed with a plastic clamp or with laboratory film. (4) The
sample is then extracted with 50 .mu.L 2M sodium acetate at pH 4.0
and 600 .mu.L of phenol/chloroform/isoamyl alcohol (10:1.93:0.036),
prepared fresh by mixing 18 mL phenol with 3.6 mL of a 1:49 isoamyl
alcohol:chloroform solution. The solution is shaken vigorously for
about 10 seconds then cooled on ice for about 15 minutes. (5) The
solution is centrifuged for about 7 minutes at maximum speed. The
upper (aqueous) phase is transferred to a new tube. (6) The RNA is
precipitated with about 10 .mu.L glycogen and with 400 .mu.L
isopropanol for 30 minutes at -20.degree. C. (7) The RNA is
pelleted by centrifugation for about 7 minutes in a benchtop
centrifuge at maximum speed; the supernatant is decanted and
discarded; and the pellet washed with approximately 500 .mu.L of
about 70 to 75% ethanol. (8) The sample is centrifuged again for 7
minutes at maximum speed. The supernatant is decanted and the
pellet air dried. The pellet is then dissolved in an appropriate
buffer for further experiments (e.g., 50 pI. 5 mM Tris chloride, pH
8.0).
Example 2
mRNA Reverse Transcription and PCR
Reverse Transcription: RNA was isolated from microdissected or
non-microdissected formalin fixed paraffin embedded (FPE) tissue as
illustrated in Example 1 and as previously described in U.S.
application Ser. No. 09/469,338 filed Dec. 20, 1999, which is
hereby incorporated by reference in its entirety. After
precipitation with ethanol and centrifugation, the RNA pellet was
dissolved in 50 ul of 5 mM Tris/Cl at pH 8.0. M-MLV Reverse
Transcriptase will extend an oligonucleotide primer hybridized to a
single-stranded RNA or DNA template in the presence of
deoxynucleotides, producing a complementary strand. The resulting
RNA was reverse transcribed with random hexamers and M-MLV Reverse
Transcriptase from Life Technologies. The reverse transcription was
accomplished by mixing 25 .mu.l of the RNA solution with 25.5 .mu.l
of "reverse transcription mix" (see below). The reaction was placed
in a thermocycler for 8 min at 26.degree. C. (for binding the
random hexamers to RNA), 45 min at 42.degree. C. (for the M-MLV
reverse transcription enzymatic reaction) and 5 min at 95.degree.
C. (for heat inactivation of DNAse).
"Reverse transcription mix" consists of 10 ul 5.times.buffer (250
mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 0.5 ul random
hexamers (50 O.D. dissolved in 550 ul of 10 mM Tris-HCl pH 7.5) 5
ul 10 mM dNTPs (dATP, dGTP, dCTP and dTTP), 5 ul 0.1 M DTT, 1.25 ul
BSA (3 mg/ml in 10 mM Tris-HCL, pH 7.5), 1.25 ul RNA Guard 24,800
U/ml (RNAse inhibitor) (Porcine #27-0816, Amersham Pharmacia) and
2.5 ul MMLV 200 U/ul (Life Tech Cat #28025-02).
Final concentrations of reaction components are: 50 mM Tris-HCl, pH
8.3, 75 mM KCl, 3 mM MgCl2, 1.0 mM dNTP, 1.0 mM DTT, 0.00375. mg/ml
BSA, 0.62 U/ul RNA Guard and 10 U/ul MMLV.
PCR Quantification of mRNA expression. Quantification of ERCC1 cDNA
and an internal control or house keeping gene (e.g., .beta.-actin)
cDNA was done using a fluorescence based real-time detection method
(ABI PRISM 7700 or 7900 Sequence Detection System [TaqMan.RTM.],
Applied Biosystems, Foster City, Calif.) as described by Heid et
al., (Genome Res 1996;6:986-994); Gibson et al., (Genome Res
1996;6:995-1001). In brief, this method uses a dual labelled
fluorogenic TaqMan.RTM. oligonucleotide probe, (ERCC1-530Tc (SEQ ID
NO: 3), T.sub.m =70.degree. C.), that anneals specifically within
the forward and reverse primers. Laser stimulation within the
capped wells containing the reaction mixture causes emission of a
3' quencher dye (TAMRA) until the probe is cleaved by the 5' to 3'
nuclease activity of the DNA polymerase during PCR extension,
causing release of a 5' reporter dye (6FAM). Production of an
amplicon thus causes emission of a fluorescent signal that is
detected by the TaqMan.RTM.'s CCD (charge-coupled device) detection
camera, and the amount of signal produced at a threshold cycle
within the purely exponential phase of the PCR reaction reflects
the starting copy number of the sequence of interest. Comparison of
the starting copy number of the sequence of interest with the
starting copy number of theinternal control gene provides a
relative gene expression level. TaqMan.RTM. analyses yield values
that are expressed as ratios between two absolute measurements
(gene of interest/internal control gene).
The PCR reaction mixture consisted 0.5 .mu.l of the reverse
transcription reaction containing the cDNA prepared as described
above 600 nM of each oligonucleoride primer (ERCC1-504F (SEQ ID NO:
1), T.sub.m =59.degree. C. and ERCC1-574R (SEQ ID NO: 2), T.sub.m
=58.degree. C.), 200 nM TaqMan.RTM. probe (SEQ ID NO:3), 5 U
AmpliTaq Gold Polymerase, 200 .mu.M each dATP, dCTP, dGTP, 400
.mu.M dTTP, 5.5 mM MgCl.sub.2, and 1.times.TaqMan.RTM. Buffer A
containing a reference dye, to a final volume of less than or equal
to 25 .mu.l (all reagents Applied Biosystems, Foster City, Calif.).
Cycling conditions were, 95.degree. C. for 10 min, followed by 45
cycles at 95.degree. C. for 15s and 60.degree. C. for 1 min.
Oligonucleotides used to quantify internal control gene
.beta.-Actin were .beta.-Actin TaqMan.RTM. probe (SEQ ID NO: 4),
.beta.-Actin-592F (SEQ ID NO: 5) and .beta.-Actin-651R (SEQ ID NO:
6).
The oligonucleotide primers ERCC1-504F (SEQ ID NO: 1) and
ERCC1-574R (SEQ ID NO: 2), used in the above described reaction
will amplify a 71 bp product.
Example 3
Determining the Uncorrected Gene Expression (UGE) for ERCC1
Two pairs of parallel reactions are carried out, i.e., "test"
reactions and the "calibration" reactions. The ERCC1 amplification
reaction and the .beta.-actin internal control amplification
reaction are the test reactions. Separate ERCC1 and .beta.-actin
amplification reactions are performed on the calibrator RNA
template and are referred to as the calibration reactions. The
TaqMan.RTM. instrument will yield four different cycle threshold
(Ct) values: Ct.sub.ERCC1 and Ct.sub..beta.-actin from the test
reactions and Ct.sub.ERCC1 and Ct.sub..beta.-actin from the
calibration reactions. The differences in Ct values for the two
reactions are determined according to the following equation:
Next the step involves raising the number 2 to the negative
.DELTA.Ct, according to the following equations.
In order to then obtain an uncorrected gene expression for ERCC1
from the TaqMan.RTM. instrument the following calculation is
carried out:
Normalizing UGE with known relative ERCC1 expression levels
The normalization calculation entails a multiplication of the UGE
with a correction factor (K.sub.ERCC1) specific to ERCC1 and a
particular calibrator RNA. A correction factor K.sub.ERCC1 can also
be determined for any internal control gene and any accurately
pre-quantified calibrator RNA. Preferably, the internal control
gene .beta.-actin and the accurately pre-quantified calibrator RNA
Human Liver Total RNA (Stratagene, Cat. #735017), are used. Given
these reagents correction factor K.sub.ERCC1 equals
1.54.times.10.sup.-3.
Normalization is accomplished using a modification of the .DELTA.Ct
method described by Applied Biosystems, the TaqMan.RTM.
manufacturer, in User Bulletin #2 and described above. To carry out
this procedure, the UGE of 6 different test tissues was analyzed
for ERCC1 expression using the TaqMan.RTM. methodology described
above. The internal control gene .beta.-actin and the calibrator
RNA,Human Liver Total RNA (Stratagene, Cat. #735017) was used.
The known relative ERCC1 expression level of each sample AG221,
AG222, AG252, Adult Lung, PC3, AdCol was divided by its
corresponding TaqMan.RTM. derived UGE to yield an unaveraged
correction factor K.
Next, all of the K values are averaged to determine a single
K.sub.ERCC1 correction factor specific for ERCC1, Human Liver Total
RNA (Stratagene, Cat. #735017) from calibrator RNA and
.beta.-actin.
Therefore, to determine the Corrected Relative ERCC1 Expression in
an unknown tissue sample on a scale that is consistent with
pre-TaqMan.RTM. ERCC1 expression studies, one merely multiplies the
uncorrected gene expression data (UGE) derived from the TaqMan.RTM.
apparatus with the K.sub.ERCC1 specific correction factor, given
the use of the same internal control gene and calibrator RNA.
A K.sub.ERCC1 may be determined using any accurately pre-quantified
calibrator RNA or internal control gene. Future sources of
accurately pre-quantified RNA can be calibrated to samples with
known relative ERCC1 expression levels as described in the method
above or may now be calibrated against a previously calibrated
calibrator RNA such as Human Liver Total RNA (Stratagene, Cat.
#735017) described above.
For example, if a subsequent K.sub.ERCC1 is determined for a
different internal control gene and/or a different calibrator RNA,
one must calibrate both the internal control gene and the
calibrator RNA to tissue samples for which ERCC1 expression levels
relative to that particular internal control gene have already been
determined. Such a determination can be made using standard
pre-TaqMan.RTM., quantitative RT-PCR techniques well known in the
art. The known expression levels for these samples will be divided
by their corresponding UGE levels to determine a K for that sample.
K values are then averaged depending on the number of known samples
to determine a new K.sub.ERCC1 specific to the different internal
control gene and/or calibrator RNA.
Example 4
ERCC1 Expression Correlates with Tumor Response
Response and survival are correlated with the level of ERCC1
expression in 56 patients with advanced non-small-cell lung cancer
(NSCLC) treated with Gem 1200 mg/m.sup.2 on days 1 and 8 plus DDP
100 mg/m.sup.2 on day 1 every 3 weeks. Patient characteristics
were: median age=59 years (range, 32-75 years); sex: 48 of the 56
patients were male (85.7%); PS=performance status (ECOG
modification of Karnofsky traditional performance status scale:
0=fully active, able to carry on all predisease performance without
restriction, 1=restricted in physically strenuous activity but
ambulatory and able to carry out work of a light or sedentary
nature): 48 patients (85.7%) were PS 0 or 1; weight loss: occurred
in 21 patients; 40 patients (71% of the total number of patients)
had Stage 4 distant metastatic disease. Median number of cycles: 3
(range 1-6).
Total mRNA was isolated from microdissected FPE pretreatment tumor
samples, and Corrected Relative ERCC1 Expression was measured using
quantitative RT-PCR as described in Examples 2 and 3. A method for
mRNA isolation from such samples is described in Example 1 and in
U.S. patent application Ser. No. 09/469,338, filed Dec. 20, 1999,
and is hereby incorporated by reference in its entirety.
Using a threshold value of 4.5.times.10.sup.-3, 35/56 patients had
high ERCC1 expression relative to internal control expression of
gene .beta.-actin. There were no differences in ERCC1 levels by
gender, age, performance status, weight loss, stage, histology or
number of cycles received. Overall response rate to the
chemotherapeutic regimen was 46.7%. The response rate was
significantly higher for 21 patients with low ERCC1 expression,
i.e. <4.5.times.10.sup.-3 (57.9%) than for 35 patients with high
ERCC1 expression, i.e. >4.5.times.10.sup.-3 (37%) (P=0.03).
Overall median survival was 32.7 weeks (range, 24.8-40.6). These
show that ERCC1 expression is a prognostic factor for response to
DDP/Gem in advanced NSCLC.
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* * * * *